In many cell lines, cyclic adenosine monophosphate, cAMP, is known to have a regulatory effect on cell proliferation and cell function. Early studies by Tomkins and colleagues as reported in Proc. Natl. Acad. Sci. USA, Vol. 70, pp. 76-79 (1973) and Am. J. Pathol. Vol. 81, pp. 199-204 (1975), have shown that, using an S49 mouse lymphoma cell line, cAMP induces these cells to undergo reversible G1 arrest, followed by cytolysis. Mutants resistant to cAMP-induced death were deficient in cAMP-dependent protein kinase, indicating that this enzyme functions in cAMP-induced cytolysis. More recent studies culminating with Lomo et al, J. ImmunoL, Vol. 154, pp. 1634-1643 (1995), have shown that the death induced by cAMP is apoptotic cell death, and occurs in normal as well as transformed lymphoid cells.
Since the first report in 1958 of an enzymatic activity capable of hydrolyzing cAMP, it has become clear that this enzymatic activity, termed cyclic nucleotide phosphodiesterase (PDE), consists of a complex isozymic superfamily represented by different forms, of which more than thirty have been identified and cloned. These isozymic PDE forms have been grouped into seven broad gene families based upon similar structural and functional relationships: Ca.sup.2+ --calmodulin-dependent (PDE1), cyclic guanosine monophosphate, cGMP, stimulated (PDE2), cGMP inhibited (PDE3), cAMP specific (PDE4), cGMP specific (PDE5), photoreceptor (PDE6), and higher affinity drug-resistant cAMP specific (PDE7). A number of reviews have been written that describe the characteristics of these different PDE forms, their regulation, potential physiological function, and progress in development of pharmacological inhibitors of PDE as therapeutic agents. Gene family-specific inhibitors have been found for all but the PDE7 gene family, but no pharmacological inhibitor is yet capable of selectively inhibiting a specific PDE isoform within a given gene family. It is believed that selective elevation of cAMP levels in transformed lymphocytes could provide a means to selectively induce apoptosis in these cells. One means of elevating cAMP levels in cells is through the inhibition of cyclic nucleotide phosphodiesterase (PDE) activity. Early studies showed that PDE activity is greatly increased in actively growing and transformed lymphocytes, that PDE activity is induced in human peripheral blood lymphocytes (HPBL) following mitogenic stimulation, and that PDE inhibitors profoundly inhibit mitogenic stimulation of HPBL. Thus, while increased PDE activity is shown in growing, cultured lymphoblastoid and leukemic cells, relative to normal, resting HPBL and long term induction of PDE activity is shown to occur in HPBL following mitogenic stimulation, the specific PDE isozyme(s) induced in HBPL were not fully characterized. Initial characterizations of PDE in HPBL suggested it was comprised mainly of PDE4 activity, and recent cloning analysis shows expression of PDE4 mRNA in HPBL. More recent biochemical analysis of PDE in purified human T lymphocytes using ion exchange HPLC separation and in HPBL by sensitivity to selective PDE inhibitors gives evidence for the presence in these cells of PDE3 as well as PDE4. The presence of PDE1 activity in a human B lymphoblastoid cell line isolated from a patient with acute lymphocytic leukemia has been documented, and it has been shown that PDE 1 activity is absent in normal, resting HPBL. Using bovine peripheral blood lymphocytes, PBL, investigators have confirmed an absence of PDE 1 activity in resting PBL and showed its appearance in these cells following mitogenic stimulation. Characterization with monoclonal antibodies suggested that the induced PDE1 activity in bovine PBL belongs to the PDE1B, 63kDa Ca.sup.2+ --calmodulin-dependent PDE, gene family.
The cDNA for PDE1B1 has been cloned from bovine, rat and mouse brain cDNA libraries. The expression of the mRNA for PDE1B1 in different tissues as assessed by Northern analysis has shown it to be restricted largely to the brain, where it is enriched in the striatum. In brain, PDE1B1 mRNA is expressed as a single species of .sup..about. 3-4 kb, whereas in mouse S49 cells three transcripts are seen at 4.4, 7, and 12 kb.
In addition to the many negative regulatory effects of cAMP, there is evidence that cAMP may play a biphasic role, being both a positive and negative regulator of lymphocyte proliferation since both mouse and human lymphocytes exhibit a late (10-50 hr.) surge in intracellular cAMP concentration following stimulation of proliferation by mitogens, which decreases just prior to the onset of DNA synthesis. Interruption of either the increase of this cAMP surge or prevention of its decrease, leading to sustained elevated cAMP, prevents the commencement of DNA synthesis.
Since sustained elevations of cAMP prevent DNA synthesis, for proliferation to proceed it is necessary for the stimulated lymphocyte to decrease its cAMP levels, and one mechanism by which it may do this is through the activation of PDE activity. Indeed, a late induction of PDE activity following mitogenesis has been described, and PDE inhibitors have been shown to profoundly inhibit lymphocyte mitogenesis.
It is also known that the proliferation of breast and prostate cancer cells is inhibited by cAMP, giving promise to the possibility that cAMP analogs or agents that raise intracellular levels of cAMP might be used in the treatment of those cancers. However, due to adverse side effects and the absence of selectivity progress has been slow, cAMP has profound effects on the metabolic machinery, growth regulatory properties and regulation of gene transcription in most cells of the body. Indiscriminate application or elevation of cAMP throughout would be likely to produce a whole array of effects, some of which may be highly undesirable. The goal, then, for employing cAMP therapy in cancer treatment is to come up with a way to selectively raise cAMP levels in cancer tissue.
The level of cAMP in cells is controlled by its rate of synthesis by adenylyl cyclase (AC) and its rate of degradation by cyclic nucleotide phosphodiesterases (PDEs). cAMP can readily be increased by agents in cancer cells by agents acting through stimulation of AC, such as catecholamines or prostaglandins acting through receptors coupled to AC, cholera toxin acting on Gs, or forskolin directly stimulating the AC catalytic unit, but none of these treatments is selective. If a specific form(s) of PDE can be shown to predominate in cancer cells, and not elsewhere, then selective inhibition of the PDE form should increase cAMP in cancer cells primarily, if not exclusively, and thereby avoid the adverse effects associated with earlier attempts at cAMP therapy.